The long-term objectives of this project are 1) to systematically map the protein-protein interaction network mediated by modular domains, 2) to understand the molecular mechanism and biological function of these interactions, and 3) to develop specific inhibitors against these domains as research tools and potential therapeutic agents. During this grant period, our studies will focus on the 104 soluble human Src homology 2 (SH2) domains, which control the specificity and fidelity of cell signaling by binding to specifi phosphotyrosyl (pY) proteins. SH2 domains recognize short peptide motifs in their partner proteins and the specificity is dictated by the pY residue and 2-4 residues flanking the pY. To understand the complex signaling network and the precise roles played by the SH2 domains, a useful first step is to identify all of the SH2 domain-pY protein interactions in human (or the SH2-pY interactome). This project employs a chemical/bioinformatics approach to map the SH2-pY interactome. The sequence specificity of an SH2 domain is systematically determined by screening a combinatorial peptide library and the consensus motif(s) is used to search the phosphoprotein databases to identify potential SH2-pY protein pairs. The putative interactions are subsequently validated (or rejected) by conventional cellular assays. The current grant period has the three specific aims. Specific Aim 1 is to determine the high-resolution specificit profiles for 40 remaining human SH2 domains (the other 64 domains have already been completed). A computer program will be developed to analyze the specificity data and search the ~19,000 known human pY motifs to construct an SH2-pY protein interaction map. Specific Aim 2 is to examine the biological function of a recently discovered 1:2 SH2-pY peptide complex during T cell signaling. Aim 3 is to assess the generality of the 1:2 complex among the 120 human SH2 domains and the sequence requirements for the peptide ligands. PUBLIC HEALTH RELEVANCE: SH2 domains play key roles during eukaryotic cell signaling. They are therefore attractive targets for therapeutic intervention of human diseases/conditions such as cancer, osteoporosis, allergy, asthma, and autoimmunity. In addition, a large number of SH2 domain mutations have been identified and shown to be associated with many human diseases. For example, mutations in Bruton tyrosine kinase SH2 domain cause hypogammaglobulinemia, antibody deficiency, and recurrent bacterial infections. Mutations in the N-SH2 domain of phosphatase SHP-2 cause Noonan syndrome, juvenile myelomonocytic leukemia, and multiple giant-cell lesion syndrome. This project will investigate the molecular mechanisms by which the SH2 domains function in physiological and pathological processes.